Effects of procollagen peptides on the translation of type II collagen messenger ribonucleic acid and on collagen biosynthesis in chondrocytes

Type II procollagen messenger ribonucleic acid (mRNA) was isolated from chick sternum and rat chondrosarcoma cells and translated in a reticulocyte lysate cell-free system. A high molecular weight band was identified as type II procollagen by gel electrophoresis, collagenase digestion, and specific immunoprecipitation. The translation of type II mRNA was specifically inhibited by addition of type I procollagen amino-terminal extension peptide. When this peptide was added to the media of cultured fetal calf chondrocytes, chick sternal chondrocytes, or chick tendon fibroblasts, no inhibition of collagen synthesis was evident. These data suggest a general regulation of collagen biosynthesis by these peptides in the cell-free translation system. However, as indicated by the cell culture experiments, cellular characteristics and evolutionary divergence of animal species seem to restrict the effect of the peptides.     

The synthesis of presumptive procollagen messenger ribonucleic acid in the calvaria of the developing chick embryo

The presumptive messenger RNAs for type I procollagen were isolated from chick embryo calvaria at various stages of development. Poly(A)-containing RNA fractions from denaturing sucrose gradients directed protein synthesis in a cell-free system derived from wheat germ. Procollagen mRNA activity was detected in a region of about 26 S. Approx. 80% of the labeled proline incorporated into cell-free product was susceptible to digestion by purified bacterial collagenase. The synthesis of procollagen mRNAs was followed during development. Comparison of the in vitro labeled mRNAs from calvaria of day 12--16 embryos indicated that the 26 S component was most pronounced at day 13 and decreased progressively towards day 16. In addition, incubation of calvaria with tritiated nucleosides for 1.5--25 h revealed that 26 S mRNA was significantly labeled only after prolonged periods. The results suggest that procollagen mRNA is a relatively stable species with a prolonged half-life compared to the majority of mRNAs in this tissue.     

Generalized inhibition of cell-free translation by the amino-terminal propeptide of chick type I procollagen

Fragments of the amino-terminal propeptide of procollagen have been shown to inhibit the synthesis of procollagen in cultured cells and in a reticulocyte lysate cell-free system (for review see Timpl, R. and Glanville, R.W. (1981) Clin. Orth. Rel. Res. 158, 224-242). In this report, we show that the full-length amino-terminal propeptide of chick pro alpha1(I) chains inhibits the translation of chick tendon mRNA and rat brain mRNA in a reticulocyte lysate cell-free system. The synthesis of procollagen and non-collagenous proteins was equally affected. Inhibition was dose-dependent up to 10 microM. A similar pattern of inhibition was observed for the collagenase-resistant fragment, col 1(I).     

Regulation of procollagen synthesis during the development of chick embryo calvaria. Correlation with procollagen mRNA content

During the embryonic development of chick calvaria (membranous cranial bones), the relative rate of procollagen synthesis increased from about 12% of total protein synthesis on Day 10 to about 65% on Day 17. This increase is due to a 1.7-fold increase in the absolute rate of procollagen synthesis and a 3-fold decrease in the synthesis of noncollagenous proteins. The increase in procollagen synthesis is directly proportional to an increase in procollagen mRNA content per cell as measured either by cell-free translation or by hybridization with complementary DNA. The results indicate that translational control of procollagen mRNA does not play a substantial role during calvaria development and that the specialization in the synthesis of this protein is largely due to the loss or inactivation of mRNAs for noncollagenous proteins.     

Levels of collagen mRNA in dedifferentiating chondrocytes

Chondrocytes liberated from chick embryo sterna were maintained in monolayer cultures and allowed to dedifferentiate. mRNA was prepared from these cultures at several intervals over a total period of 6 weeks. Levels of α1(I) and α2(I) procollagen mRNA were assayed by cell-free translation and by Northern blots using cDNA clones specific for the respective procollagen chains. Dedifferentiating chondrocytes first take up synthesis of α1(I) mRNA which is followed after several days by synthesis of α2(I) mRNA. This two-step mechanism for the onset of procollagen type I mRNA synthesis is accompanied by a proceeding loss of α1(II) procollagen gene expression.     

Cortisol decreases the cellular concentration of translatable procollagen mRNA species in cultured human skin fibroblasts

The effect of cortisol on the cellular concentration of translatable procollagen mRNAs was studied in cultured human skin fibroblasts. Cortisol selectively decreased the amount of procollagen mRNAs, in comparison to the total mRNA activity, when the cells were grown in enriched medium conditions, i.e., with 10% newborn calf serum. The selective decrease was first observed after 6 h exposure to 1 microM cortisol. In depleted medium conditions, i.e., with 2% newborn calf serum, the initial response was a stimulatory one, followed after about 12 h by a decrease in the procollagen mRNA activity. The results suggest that the selective inhibitory effect of cortisol on the cellular concentration of translatable procollagen mRNA species needs an optimal serum concentration. Furthermore, the results give support to the hypothesis that the decrease in the procollagen mRNA concentration after cortisol administration is a secondary response, preceded by the induction of some intracellular regulation system.     

Cortisol decreases the cellular concentration of translatable procollagen mRNA species in cultured human skin fibroblasts

The effect of cortisol on the cellular concentration of translatable procollagen mRNAs was studied in cultured human skin fibroblasts. Cortisol selectively decreased the amount of procollagen mRNAs, in comparison to the total mRNA activity, when the cells were grown in enriched medium conditions, i.e., with 10% newborn calf serum. The selective decrease was first observed after 6 h exposure to 1 μM cortisol. In depleted medium conditions, i.e., with 2% newborn calf serum, the initial response was a stimulatory one, followed after about 12 h by a decrease in the procollagen mRNA activity. The results suggest that the selective inhibitory effect of cortisol on the cellular concentration of translatable procollagen mRNA species needs an optimal serum concentration. Furthermore, the results give support to the hypothesis that the decrease in the procollagen mRNA concentration after cortisol administration is a secondary response, preceeded by the induction of some intracellular regulation system.     

Cloned complementary deoxyribonucleic acid probes for untranslated messenger ribonucleic acid components of mouse sarcoma ascites cells

Mouse sarcoma ascites cells contain several abundant mRNA species that occur to a large extent in an untranslated state. RNA preparations enriched in these species were used as starting material to construct recombinant plasmids. Cloned plasmids bearing sequences homologous to four of the untranslated mRNA species were identified by translation of hybrid-selected material. These plasmids, as well as a recombinant plasmid derived from chick alpha-actin mRNA, were used as probes for the estimation of mRNA levels in polyribosomes and in small ribonucleoprotein (RNP) particles of the ascites cells. Considerable amounts of the mRNA molecules belonging to the untranslated species were present in polyribosomes as well as in mRNPs. The actin mRNA, on the other hand, was present almost exclusively in polyribosomes. The distributions obtained by the hybridization assay resembled those estimated by translation of the same RNA preparations in cell-free systems. This indicates that the mRNA molecules of a given species engaged in translation in the cells and those present as untranslated RNP particles are equally effective in cell-free translation systems.     

Expression of type X collagen mRNA levels in embryonic chick sternum during development

Embryonic chick sternum cartilage exhibits profound spatial and temporal changes in Type X collagen biosynthesis during development. Production of this collagen is confined to the presumptive calcification region and its expression is not acquired until stage 43. To examine the mechanisms responsible for regulation of developmental changes in biosynthetic expression of Type X collagen, we determined the levels of translatable Type X procollagen mRNA employing a cell-free translation system. We found that mRNA capable of directing Type X collagen synthesis was present exclusively in cartilage destined to undergo calcification and that its levels were nearly equivalent at all stages of development. These findings suggest that expression of Type X collagen in embryonic chick sternum is determined at the translational level.     

Glucocorticoids decrease the synthesis of type I procollagen mRNAs

Glucocorticoids selectively decrease procollagen synthesis in animal and human skin fibroblasts. beta-Actin content and beta-actin mRNA are not affected by glucocorticoid treatment of chick skin fibroblasts. The inhibitory effect of glucocorticoids on procollagen synthesis is associated with a decrease in total cellular type I procollagen mRNAs in chick skin fibroblasts. These effects of dexamethasone are receptor mediated as determined by pretreatment with the glucocorticoid antagonists progesterone and RU-486 and with the agonist beta-dihydrocortisol. Dexamethasone has a small but significant inhibitory effect on cell growth of chick skin fibroblasts. The ability of this corticosteroid to decrease the steady-state levels of type I procollagen mRNAs in nuclei, cytoplasm, and polysomes varies. The largest decrease of type I procollagen mRNAs is observed in the nuclear and cytoplasmic subcellular fractions 24 h after dexamethasone treatment. Type I procollagen hnRNAs are also decreased as determined by Northern blot analysis of total nuclear RNA. The synthesis of total cellular type I procollagen mRNAs is reversibly decreased by dexamethasone treatment. In addition the synthesis of total nuclear type I procollagen mRNA sequences is decreased at 2, 4, and 24 h following the addition of radioactive nucleoside and dexamethasone to cell cultures. Although the synthesis of pro alpha 1(I) and pro alpha 2(I) mRNAs is decreased in dexamethasone-treated chick skin fibroblasts, the degradation of the total cellular procollagen mRNAs is not altered while the degradation of total cellular RNA is stabilized. These data indicate that the dexamethasone-mediated decrease of procollagen synthesis in embryonic chick skin fibroblasts results from the regulation of procollagen gene expression.     

Translation of myosin heavy chain messenger ribonucleic acid in an eukaryotic initiation factor 3- and messenger-dependent muscle cell-free system.

A cell-free protein synthesis system has been prepared from embryonic chick muscle; this system is dependent on initiation factor eukaryotic initiation factor 3 (eIF-3) and mRNA for efficient translation. Highly purified chick muscle eIF-3 has been fractionated into "core" and discriminatory components. In the presence of core eIF-3 from chick muscle or rabbit reticulocytes, myosin heavy chain mRNA is translated less efficiently than globin mRNA present in an equimolar concentration. When the discriminatory components are added to core eIF-3 from either source, myosin mRNA is translated with a greater efficiency. Thus, chick muscle eIF-3 contains components which allow it to recognize and stimulate specifically the translation of myosin mRNA in a muscle cell-free protein synthesis system.     

Isolation and cell-free translation of chick lens crystallin mRNA during normal development and transdifferentiation of neural retina

Messenger RNA has been isolated from day-old chick lens. Size characterization and heterologous cell-free translation demonstrate that the predominant species of mRNA present code for α-, β- and δ-crystallins. Total polysomal RNA and polysomal RNA which did not bind to oligo (dT)-cellulose translate in the cell-free system to give a crystallin profile qualitatively similar to that of poly(A)⁺ mRNA. RNA from postribosomal supernatant which binds to oligo(dT)-cellulose also translates to give crystallins, but the products are enriched for β-crystallins. Messenger RNAs isolated from 15-day embryo lens fiber and lens epithelium cells give products on translation which reflect the different protein compositions of these two cell types, as do mRNAs isolated from chick lenses at various developmental stages. Messenger RNAs were isolated from freshly excised 8-day embryo neural retina and from this tissue undergoing transdifferentiation into lens cells in cell culture. Cell-free translation demonstrates no detectable crystallin mRNAs in the freshly excised material, but by 42 days in cell culture, crystallin mRNAs are the most prominent species.     

Role of FK506-binding protein 12 in development of the chick embryonic heart

A cDNA encoding chicken FK506-binding protein 12 (FKBP12) was isolated and sequenced. The predicted amino acid sequence of the chicken protein shows high homology to those of FKBP12 proteins of other species ranging from human to frog. The possible role of FKBP12 in chick embryonic cardiac development was examined. Northern blot analysis revealed that FKBP12 mRNA is distributed widely in chick embryos, being especially abundant in the heart; the amount of FKBP12 mRNA in the embryonic heart decreased with time. Administration of FK506 to chick embryos at 7 to 9 days resulted in marked cardiac enlargement. FK506 also reduced the expression of myosin, induced a more elongated cell morphology, and impaired network formation in cultured chick embryonic cardiomyocytes. These results suggest that FKBP12 is important in the regulation of contractile function and phenotypic expression in chick cardiomyocytes during embryonic development.     

Cortisol induces (2′–5′)oligoadenylate synthetase in cultured chick embryo tendon fibroblasts

The changes in the (2′–5′)oligoadenylate synthetase activity after cortisol administration were studied in chick embryo tendon fibroblasts. The enzyme activity correlated with the cortisol concentration of the growth medium. The results indicate that the decrease in the cellular concentration of some mRNA species, such as procollagen type I mRNA species, after cortisol administration may at least partly be due to activation of ribonuclease L.     

Identification of amino-acid substitutions in the proteolipid subunit of the ATP synthase from dicyclohexylcarbodiimide-resistant mutants of Escherichia coli

Polyadenylated mRNA was isolated from chick embryo liver following induction of hepatic porphyria. The RNA was translated in vitro using a wheat germ cell-free system and delta-aminolaevulinate synthase was identified in the translation products by indirect immunoprecipitation. The enzyme was not apparent in the translation products of polyadenylated RNA from non-induced livers. The molecular weight of delta-aminolaevulinate synthase synthesized in vitro was 70000 and the protein was estimated to represent up to 5% of total products synthesised in vitro. These data demonstrate for the first time that induction of chick embryo liver delta-aminolaevulinate synthase activity in hepatic porphyria correlates with a large increase in the translational capacity of isolated polyadenylated RNA for this enzyme and, together with preliminary cDNA . RNA hybridization studies, indicate that an increase in the level of delta-aminolaevulinic synthase mRNA is responsible.     

Rapid disappearance of translatable actin mRNA during cell differentiation in Naegleria

Actin, the major protein of Naegleria gruberi, is selectively not synthesized during the differentiation of amebae to flagellates. When RNA extracted from cells at intervals during differentiation is translated in the wheat germ cell-free system, a major translation product with the electrophoretic mobility of actin is seen to disappear with time during differentiation. This translation product is shown to be actin by its electrophoretic mobility, copolymerization with rabbit actin, peptide map, and immunoprecipitation by antibodies specific to Naegleria actin. Multiple isoforms of actin are synthesized in the cell-free system. Quantitative immunoprecipitation of translation products was employed to measure the relative amount of actin mRNA. Translatable actin mRNA begins to decrease in abundance within 7 min after the initiation of differentiation and thereafter decreases with a half-life of about 25 min. The selective disappearance of this major translatable mRNA provides a favorable opportunity to dissect the rules governing the half-life of a specific mRNA.     

Procollagen mRNA metabolism during the fibroblast cell cycle and its synthesis in transformed cells.

Procollagen mRNA was isolated from mouse embryos and used for the synthesis of a highly labelled cDNA probe complementary to collagen mRNA. This probe was used for the investigation of procollagen mRNA metabolism during the cell cycle of 3T6 mouse embryo fibroblasts in culture. Titration hybridization experiments revealed that procollagen mRNA was present throughout the cell cycle following stumulation of confluent monolayers. Procollagen mRNA levels of sparse cultures appeared similar to those of unstimulated monolayers. The fluctuating levels of collagen synthesis during the cell cycle can be ascribed to changes in the amount of collagen mRNA present. In mouse sarcoma virus transformed 3T3 cells only 20--30% of the amount of procollagen mRNA in 3T3 cells is present indicating that the decline in collagen synthesis is due to mRNA availability.